The goal of this research proposal is to understand the role of protein modification by the ubiquitin-like molecule ISG15, using the target protein phospholipase C gamma 1 as a model. ISG15 is one of the first and one of the most abundant genes upregulated following interferon treatment. ISG15 can be detected as a free protein or in conjugates with other proteins. ISG15 has been identified only in vertebrates, indicating that is has a function specific to higher eukaryotes. Increased protein modification by ISG15 has been directly linked to the hypersensitivity of interferon treatment. However, the molecular mechanism of protein ISG15 modification is largely unknown. The specific aims of this proposal are to use phospholipase C gamma 1 as a model to localize the ISG15 modification site, to identify the role of ISG15 modification in protein cellular localization, and to analyze its effect on protein function.